Emerging evidence suggests that cancer progression is often associated with the acquisition of Epithelial-to- Mesenchymal Transition (EMT) phenotype that is reminiscent of "Cancer Stem-like Cells (CSCs)", which is partly responsible for the ability of pancreatic cancer (PC) cells to acquire aggressiveness, contributing to tumor metastasis. Our published data showed decreased expression of miR-200 in gemcitabine resistant EMT- type cells, resulting in the up-regulation of ZEB1 and thereby down-regulation of E-cadherin, and also showed increased expression of miR-21, resulting in the inactivation of PTEN expression. We also found significantly higher levels of miR-21 in the plasma of PC patients compared to normal volunteers, which was correlated with worsened survival. Based on these results, we reasoned that finding a novel non-toxic avenue by which miR- 200 could be up-regulated and miR-21 could be down-regulated would be a novel approach for the prevention of tumor progression. To that end, we have published, for the first time, that a synthetic analog of a non-toxic natural agent curcumin, named CDF is superior in its target tissue (pancreas) bioavailability without any adverse side effects, and the CDF was very effective in the elimination of EMT phenotypic cells, which was in part due to up-regulation of miR-200 and down-regulation of miR-21, resulting in the down-regulation of ZEB1, and increased expression of E-cadherin and PTEN. Therefore, it appears that we found a novel approach by which "conditioning" of the biological milieu of drug-resistant EMT-type cells could be achieved toward effective elimination of EMT-type cells or CSCs, which could be useful for the prevention of tumor progression by eliminating the "root" cause of tumor recurrence. Based on our preliminary results, we hypothesize that the activation of miR-21 is critical during the acquisition of EMT phenotype in gemcitabine-resistant (GR) PC cells, and these cells could be eliminated by CDF alone or in combination with conventional therapeutics. We will test our hypothesis to gain mechanistic insight on the role of miR-21 and PTEN during the acquisition of EMT phenotype in GR cells, and investigate the biological consequence of these cells by manipulating the expression of miR-21 (Aim-1). We will also investigate how CDF could down-regulate miR- 21 and up-regulate the expression of its targets. This may lead to the "conditioning" of the biological milieu of PC cells, resulting in sensitization of cells to conventional agents (Aim-2;in vitro studies). Finally, we will determine whether CDF could cause increased anti-tumor activity when combined with conventional therapeutics in vivo using xenograft and K-ras transgenic animal models compared to CDF alone (Aim-3). The results of our studies will provide mechanistic insight as to the role of miR-21 in drug-resistant EMT-type cells (CSCs), and will also provide pre-clinical data in support of the role of CDF for the prevention of tumor progression and/or treatment of PC. Therefore, our results will have significantly high impact toward preventing tumor recurrence, which will lead to achieve better survival outcome of patients diagnosed with PC. PUBLIC HEALTH RELEVANCE: This project is focused on elucidating the mechanism of Epithelial-to-Mesenchymal Transition (EMT) in gemcitabine-resistant (GR) pancreatic cancer (PC) cells related to the regulation of miR-21 and its targets, and further investigate the mechanism(s) by which "natural agents" such as curcumin and our novel synthetic analog (CDF) of curcumin could effectively kill EMT-type cells alone or in combination with conventional chemotherapeutic agents. We will examine the molecular mechanism in vitro and will also test our hypothesis in vivo using xenograft and K-ras transgenic animal model by assessing whether CDF or curcumin alone or in combination with conventional therapeutics could be useful for the prevention of tumor progression and/or treatment. Our proposed investigation is highly relevant to the mission of NCI, NIH and is likely to have a significant impact toward saving lives of patients diagnosed with PC in the immediate future.